The basic principle that distinguishes a confocal microscope from other forms of light microscopy is that discrete aperture spots are illuminated in the object plane of the microscope from which reflected or fluorescent light is then relayed for observation through conjugate apertures in the image plane. For diffraction limited spots, this principle results in spacial resolution 1.4 times better than the optimum resolution obtainable by conventional light microscopy. Furthermore, this method dramatically reduces the interference of stray, out-of-focus light from an observed specimen above or below the focal plane. This technique actually permits optical sectioning of living tissue (with depth restrictions) and high-resolution 3-D reconstruction with automated focussing controls. It is the only microscopic technique that can effectively resolve individual cells in living tissue without staining.
Presently known confocal microscopes employ a sequential scanning technique which involves scanning each illumination and image conjugate aperture pair one at a time until the object (and image) has been covered sufficiently to produce an adequate confocal image. A basic form of the confocal microscope is disclosed in U.S. Pat. No. 3,013,467 to Minsky.
Scanning technologies which have been developed for known confocal microscopes include the mechanical translation of the specimen with fixed optics, but scanning has also been accomplished using a fixed specimen and scanning beams manipulated by special rotating aperture disks. The use of such rotating disks is disclosed in U.S. Pat. Nos. 4,802,748 to McCarthy et al., 5,067,805 to Corle et al., 5,099,363 to Lichtman and 5,162,941 to Favro et al. Although these disks include a plurality of apertures, only one aperture at a time is used for scanning. Other known confocal scanning systems have included a laser beam rastered with rotating mirrors to scan a specimen, or a laser beam which scans a slit rather than a spot. Such slit scanning increases imaging speed but slightly degrades resolution.
Conventional confocal microscopes are subject to a number of limitations imposed by the scanning techniques employed. In some cases, confocal image acquisition is too slow for certain applications and becomes slower as the scan line density decreases and as aperture separation increases. Furthermore, these parameters are preset and are not practically adjustable in most commercial systems. Signal-to-noise ratio (SNR) must be severely sacrificed to increase imaging rate. Trying to improve SNR by increasing illumination intensity is often limited by the susceptibility of live cells to photodamage or rapid fluorophore photobleaching.
For most conventional confocal systems, proper alignment is critical and may be difficult to maintain. The same physical aperture must be used for illumination and detection thereby limiting this technique to the study of reflection and epifluorescence. Therefore, transmitted light confocal imaging through a translucent slice (as for many voltage-sensitive dyes) is not practically possible.
Finally, it is often difficult or impossible for the user to restrict illumination and imaging to any selected subfield and differentially control light exposure.
Laser scanning systems have become widely accepted because of their higher intensity and diffraction limited spot size which results in greater resolution in the confocal image. However, they are more expensive than white-light systems and do not offer the selection of illumination wavelengths needed for the growing varieties of fluorescence indicators now available. Furthermore, laser intensity often leads to problems with phototoxicity and rapid photobleaching.
Confocal imaging as now known has several significant physical constraints. Because only a small portion (e.g. 1%) of light from a light source is actually used for imaging, light efficiency is challenged in multiple point source scanning confocal imaging. The requirement of a high power light source to compensate for the low light efficiency in confocal imaging results in the generation of heat necessitating the use of cooling fans in light modules which generate harmful vibration.
There exist many fluorescent indicators which operate in the dual excitation, single emission ratio mode; for example, Fura-2 and BTC for calcium and BCECF for pH. Dual excitation indicators have proven to be problematic in ratio imaging microscopy because the two images acquired with different excitation wavelengths can never be obtained simultaneously. Reliable ratio imaging requires that the temporal separation of the two images be minimized relative to the rate of change of the ligand species which binds to the fluorescent indicator. Products for switching between two excitation sources include filter wheels and other mechanical devices (shutters, oscillating filters, etc.) and acousto-optics modulators or tunable filters. Mechanical devices are slow and may introduce vibrations which are detrimental to imaging. Although acousto-opticals are fast, solid state devices, they are extremely inefficient since they operate as diffraction gratings; only a small fraction of the incident light is available for excitation.
The fundamental limitation for image acquisition speed is the number of fluorescent photons detected. Rapid switching between two excitation sources at speeds greater than that required to produce the number of fluorescence photons needed for a given signal to noise figure of merit is not useful. Many of the fluorescent indicators have different overall fluorescence efficiencies for the ligand bound and free indicator compound. Thus there is a need for the ability to maximize the signal to noise properties of ratio imaging by providing variable dwell time for the bound and free indicator compound. Then the total number of photons needed for a given signal to noise figure of merit can be achieved with both images used to form the ratio by compensating for differing fluorescence efficiencies with variable excitation exposure times.